The clinical amelioration in sickle cell disease seen with persistent fetal hemoglobin expression suggests that treatment regimens capable of reversing ontogeny and re-activating gamma-globin gene expression would be explored for this disease. Gene transfer of a specific transcription factor which will retard or reverse the gamma/beta-globin switch provides one approach to this aim. An advantage of this strategy over the, as yet unsuccessful, transfer of globin genes, is the concomitant reduction in beta(s)- expression that would be anticipated. The long-term objective of this proposal is to identify a factor(s) which may be utilized in this approach to gene therapy. One candidate is CP2, a protein that has recently been identified as a component of the human stage selector protein complex (SSP). The studies proposed in the first specific aim seek to define the role of CP2 in the formation of the SSP complex and to determine the mechanisms by which it confers developmental specificity on the gamma-promoter. Definition of the protein/protein interactions of this factor, studies of its developmental expression, and functional analysis of its interaction with the LCR will determine how CP2 modifies gamma-gene expression. In the second specific aim, another stage selector sequence, recently localized within the proximal gamma-promoter, will be characterized and the proteins mediating its function identified. These proteins may provide additional candidate factors for evaluation in the model systems detailed in project 4. Collectively, these studies will enhance our understanding of the basic molecular regulation of globin gene expression and may provide new therapeutic strategies for the treatment of SCD.